Difference between revisions of "Part:BBa K2980002"
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<partinfo>BBa_K2980002 parameters</partinfo>
 
<partinfo>BBa_K2980002 parameters</partinfo>
 
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==Contribution: NUDT_CHINA 2021==
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In our project for iGEM2021, we use this basic part with CRY2([[Part:BBa_K2980000]]) to fuse with other proteins so as to introduce a blue light-induced switch to our protein degradation system. However, we find this part document lack experimental validation to prove its interaction with CIB1 under blue light stimulation. Therefore, we constructed plasmids based on the mechanism of tet-on system, co-transfected the plasmids into HEK293T cells, applied the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48h and then conducted SEAP assay to validate the interaction between this part and CIB1.
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===Method===
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We separately expressed tetR fused with CIB1 and CRY2 fused with VP64 so that only by applying blue light stimulus would the two proteins bind to each other through CRY2-CIB1 interaction.Meanwhile, we utilized a translational control element (TCE) which consists of seven tetO to enable the transcription of reporter gene (SEAP) downstream once bound with tetR and VP64. Under this assumption, the expression level of SEAP can illustrate whether CRY2 interacts with CIB1 under blue light and we can further demonstrate the interaction level through SEAP assay.
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<figure class="figure">
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<img src="https://static.igem.org/mediawiki/parts/8/8e/T--NUDT_CHINA--Part_Validation_SEAP_CRY2-CIB1.png" class="figure-img img-fluid rounded"  height="350px">
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</figure>
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</html>
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Figure1. Experimental validation approach of CRY2 and CIB1
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*Here is the protocol for SEAP assay:
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1.Cell transfection and stimulation
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(1)Seed HEK293T cells into 24-well cell culture plates. (Approximately 5  cells per well)
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(2)Culture for 16 h before transfection
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(3)Total plasmid mixes of 800ng per well are mixed thoroughly in DMEM before a polyethylenimine (PEI) solution (1 mg/ml) is added into the plasmid mixture in a ratio of 1:5 (plasmid weight/PEI weight)
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(4)The plasmid–PEI mixture is vortexed and incubated at room temperature for 15 min. The mixture is then added into the cells and incubated for at least 6 h.
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(5)Cells are then changed into fresh medium and apply the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48 h before sampling and analysis assay. Culture the control group in the dark for the same period.
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2.SEAP assay in vitro
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(1)Sample 200 ul culture medium from each well, heat inactivate at 65 ℃ for 30 min
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(2)During the heat inactivation procedure, warm up 2 SEAP buffer (100 ul/well) at 37 ℃
 +
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(3)Add 1/5 buffer volume of pNPP (20 μL/well) substrate into the 2x buffer to prepare the “Detection Mixture.”
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(4)Measure absorption at 405 nm, 37 s per read for 10 reads.
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(5)Calculate enzymatic activity.
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===Result===
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<html>
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<figure class="figure">
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<img src="https://2021.igem.org/wiki/images/8/89/T--NUDT_CHINA--Contribution_CRY2-CIB1_.png
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" class="figure-img img-fluid rounded"  height="350px">
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</figure>
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</html>
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Figure2. SEAP activity of CRY2-CIB1 under dark and 0/24/48/72h cell culturing with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval).
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The interaction between the two proteins can be detected by SEAP analysis. The result showed a significantly high SEAP activity of the experimental group compared to the control group, proving the interaction with CRY2 and CIB1 under blue light can be observed.

Revision as of 05:46, 21 October 2021


CIB1

CIB1 will interact with CRY2 (Part:BBa K2980000) under blue light stimulation. It is the main light control element in our system.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 112
  • 1000
    COMPATIBLE WITH RFC[1000]



Contribution: NUDT_CHINA 2021

In our project for iGEM2021, we use this basic part with CRY2(Part:BBa_K2980000) to fuse with other proteins so as to introduce a blue light-induced switch to our protein degradation system. However, we find this part document lack experimental validation to prove its interaction with CIB1 under blue light stimulation. Therefore, we constructed plasmids based on the mechanism of tet-on system, co-transfected the plasmids into HEK293T cells, applied the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48h and then conducted SEAP assay to validate the interaction between this part and CIB1.

Method

We separately expressed tetR fused with CIB1 and CRY2 fused with VP64 so that only by applying blue light stimulus would the two proteins bind to each other through CRY2-CIB1 interaction.Meanwhile, we utilized a translational control element (TCE) which consists of seven tetO to enable the transcription of reporter gene (SEAP) downstream once bound with tetR and VP64. Under this assumption, the expression level of SEAP can illustrate whether CRY2 interacts with CIB1 under blue light and we can further demonstrate the interaction level through SEAP assay.

Figure1. Experimental validation approach of CRY2 and CIB1


  • Here is the protocol for SEAP assay:

1.Cell transfection and stimulation

(1)Seed HEK293T cells into 24-well cell culture plates. (Approximately 5 cells per well)

(2)Culture for 16 h before transfection

(3)Total plasmid mixes of 800ng per well are mixed thoroughly in DMEM before a polyethylenimine (PEI) solution (1 mg/ml) is added into the plasmid mixture in a ratio of 1:5 (plasmid weight/PEI weight)

(4)The plasmid–PEI mixture is vortexed and incubated at room temperature for 15 min. The mixture is then added into the cells and incubated for at least 6 h.

(5)Cells are then changed into fresh medium and apply the experiment group with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval) for 24/48 h before sampling and analysis assay. Culture the control group in the dark for the same period.


2.SEAP assay in vitro

(1)Sample 200 ul culture medium from each well, heat inactivate at 65 ℃ for 30 min

(2)During the heat inactivation procedure, warm up 2 SEAP buffer (100 ul/well) at 37 ℃

(3)Add 1/5 buffer volume of pNPP (20 μL/well) substrate into the 2x buffer to prepare the “Detection Mixture.”

(4)Measure absorption at 405 nm, 37 s per read for 10 reads.

(5)Calculate enzymatic activity.


Result

Figure2. SEAP activity of CRY2-CIB1 under dark and 0/24/48/72h cell culturing with blue light stimulus (480nm, stimulate 2 seconds with a 58 second-interval).

The interaction between the two proteins can be detected by SEAP analysis. The result showed a significantly high SEAP activity of the experimental group compared to the control group, proving the interaction with CRY2 and CIB1 under blue light can be observed.